Method and composition for the modulation of angiogenesis

ABSTRACT

We have surprisingly discovered that the enzyme glucose-6-phosphate dehydrogenase (G6PD) regulates angiogenesis. As a result of this discovery, the present invention provides modulation of angiogenesis in tissues where that angiogenesis depends upon the activity of G6PD.

This invention was made in part with U.S. Government support underContact Numbers P01 HL55993 and HL04399 awarded by the NationalInstitutes of Health. The U.S. Government has certain rights in thisapplication.

FIELD OF THE INVENTION

The present invention provides for novel pharmaceutical compositions,and methods of use thereof for treatment of diseases or disordersinvolving angiogenesis.

BACKGROUND OF THE INVENTION

Blood vessels are the means by which oxygen and nutrients are suppliedto living tissues and waste products are removed from living tissue.Angiogenesis refers to the process by which new blood vessels areformed. (1; reviewed by Folkman, J., 2001, Semin. Oncol. 28 (6): 536-42;Ribatti, D., et al., 2000, Gen. Pharmacol. 35 (5): 227-31). Thus, whereappropriate, angiogenesis is a critical biological process. It isessential in reproduction, development and wound repair. However,inappropriate angiogenesis can have severe negative consequences. Forexample, it is only after many solid tumors are vascularized as a resultof angiogenesis that the tumors have a sufficient supply of oxygen andnutrients that permit it to grow rapidly and metastasize. Becausemaintaining the rate of angiogenesis in its proper equilibrium is socritical to a range of functions, it must be carefully regulated inorder to maintain health. The angiogenesis process is believed to beginwith the degradation of the basement membrane by proteases secreted fromendothelial cells (EC) activated by mitogens such as vascularendothelial growth factor (VEGF) and basic fibroblast growth factor(bFGF). The cells migrate and proliferate, leading to the formation ofsolid endothelial cell sprouts into the stromal space, then, vascularloops are formed and capillary tubes develop with formation of tightjunctions and deposition of new basement membrane.

In adults, the proliferation rate of endothelial cells is typically lowcompared to other cell types in the body. The turnover time of thesecells can exceed one thousand days. Physiological exceptions in whichangiogenesis results in rapid proliferation typically occurs under tightregulation, such as found in the female reproductive system and duringwound healing.

The rate of angiogenesis involves a change in the local equilibriumbetween positive and negative regulators of the growth of microvessels.The therapeutic implications of angiogenic growth factors were firstdescribed by Folkman and colleagues over two decades ago (2). Abnormalangiogenesis occurs when there are increased or decreased stimuli forangiogenesis resulting in either excessive or insufficient blood vesselgrowth, respectively. For instance, conditions such as ulcers, strokes,and heart attacks may result from the absence of angiogenesis normallyrequired for natural healing. In contrast, excessive blood vesselproliferation can result in tumor growth, tumor spread, blindness,psoriasis and rheumatoid arthritis.

Thus, there are instances where a greater degree of angiogenesis isdesirable—increasing blood circulation, wound healing, and ulcerhealing. For example, investigations have established the feasibility ofusing recombinant angiogenic growth factors, such as fibroblast growthfactor (FGF) family (3, 4), endothelial cell growth factor (ECGF) (5),and more recently, vascular endothelial growth factor (VEGF) to expediteand/or augment collateral artery development in animal models ofmyocardial and hindlimb ischemia (5, 6).

Conversely, there are instances, where inhibition of angiogenesis isdesirable. For example, many diseases are driven by persistentunregulated angiogenesis, also sometimes referred to as“neovascularization.” In arthritis, new capillary blood vessels invadethe joint and destroy cartilage. In diabetes, new capillaries invade thevitreous, bleed, and cause blindness. Ocular neovascularization is themost common cause of blindness. Tumor growth and metastasis areangiogenesis-dependent A tumor must continuously stimulate the growth ofnew capillary blood vessels for the tumor itself to grow.

The current treatment of these diseases is inadequate. Agents whichprevent continued angiogenesis, e.g, drugs (TNP470), monoclonalantibodies, antisense oligodeoxynucleotides and proteins (angiostatin(7), endostatin (8) and antiangiogenic ATIII (9)) are currently beingtested. (10, 11, 12). Although preliminary results with theantiangiogenic proteins are promising, new antiangiogenic agents thatshow improvement in size, ease of production, stability and/or potencywould be desirable.

SUMMARY OF THE INVENTION

We have surprisingly discovered that the enzyme glucose-6-phosphatedehydrogenase (G6PD) regulates angiogenesis. As a result of thisdiscovery, the present invention provides modulation of angiogenesis intissues where that angiogenesis depends upon the activity of G6PD.

The present invention further provides compositions and methods formodulating angiogenesis in a tissue associated with a disease condition.A composition comprising an angiogenesis-modulating amount of a G6PDprotein or an antagonist thereof is administered to tissue to be treatedfor a disease condition that responds to modulation of angiogenesis.

The composition providing the G6PD protein can contain purified protein,biologically active protein fragments, recombinantly produced G6PDprotein or protein fragments or fusion proteins, or gene/nucleic acidexpression vectors for expressing a G6PD protein.

The composition containing the G6PD antagonist can contain anti-G6PDantibodies, androsterone steroids, including dehydroepiandrosterone, and16α-fluro-5α-androstan-17-one (FAO), as well as 6-aminonicotinamide andnicotinamide derivatives, RNAi, antisense oligoneuleotides, for example,antisense phosphorothioate oligodeoxynucleotides(5′-AGGUCACCCGAUGCACCCAUGAUGA-3′ (SEQ ID NO: 1)), including any sequencebetween 8-30 bases in any combination that results in inhibition of G6PDgene expression by hybrid arrest of translation or ribonuclease Hdigestion of the formed RNA-DNA heteroduplex. G6PD antagonists furtherinclude adenoviral-mediated overexpression of dominant negative forms ofG6PD or G6PD cDNA with reduced activity. Proteins, muteins, peptides,mimetics and small molecule drugs that inhibit the angiogenic activityof G6PD can also be used.

The present invention can be used alone or in combination with otherstrategies to modulate angiogenesis.

Where the G6PD protein is inactivated or inhibited, the modulation is aninhibition of angiogenesis. Where the G6PD protein is active oractivated, the modulation is a potentiation of angiogenesis. The tissueto be treated can be any tissue in which modulation of angiogenesis isdesirable. For angiogenesis inhibition, it is useful to treat diseasedtissue where deleterious neovascularization is occurring. Exemplarytissues include, solid tumors, metastases, tissues undergoingrestenosis, and the like tissues. Inhibition of angiogenesis would alsobe beneficial in disease states characterized by pathologicalangiogenesis, such as tumor growth, diabetic retinopathy, vascularrestenosis, primary pulmonary hypertension, hereditary hemorrhagictelangiectasis, post-operative adhesion formation, atherosclerosis andrheumatoid arthritis.

For potentiation, it is useful to treat patients with hypoxic tissuessuch as those following stroke, myocardial infarction or associated withchronic ulcers, tissues in patients with ischemic limbs in which thereis abnormal, i.e., poor circulation, due to diabetic or otherconditions. Patients with chronic wounds that do not heal, and thereforecould benefit from the increase in vascular cell proliferation andneovascularization, can be treated as well. Potentiation of angiogenesiswould also offer therapeutic benefit for ischemic vascular diseases,including coronary artery insufficiency and ischemic cardiomyopathy,peripheral arterial occlusive disease, cerebrovascular disease, ischemicbowel syndromes, impotence, and would healing.

The G6PD protein, peptide, and nucleic acid sequence encoding G6PDprotein or peptide may be administered in conjunction with anotherangiogenesis stimulator.

The present invention also provides for methods to increase G6PDactivity by means of administration of insulin or dietary manipulation.

The present invention also encompasses a pharmaceutical compositionsuitable for stimulating angiogenesis in a target mammalian tissuecomprising a viral or non-viral gene transfer vector containg a nucleicacid, the nucleic acid having a nucleic acid segment encoding for a G6PDprotein or peptide, and a pharmaceutically acceptable carrier.

Also envisioned is a pharmaceutical composition suitable for inhibitingangiogenesis in a target mammalian tissue and comprising a viral ornon-viral gene transfer vector containing, for example, a nucleic acidhaving a segment encoding for an antisense oligonucleotide to G6PD mRNAand a pharmaceutically acceptable carrier.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show G6PD and endothelial cell proliferation. BAEC weretreated with DHEA to decrease G6PD activity and stimulated with VEGF(100 ng/ml) for 8 hr (1A). Cell proliferation is measured by[³H]-Thymidine incorporation. G6PD expression was inhibited bytrans(in)fection with an antisense oligonucleotide to G6PD mRNA (AS) ora scrambled control (SS) and proliferation was determined by[³H]-Thymidine incorporation (1B). Results are expressed as counts perminute (cpm) and data presented as mean±SEM. *p<0.01 vs.−VEGF.**p<0.01vs.−DHEA or −SS.

FIGS. 2A and B show overexpression of G6PD using adenoviral genetransfer. G6PD expression is significantly increased followingtrans(in)fection with pAD-G6PD (2.5×MOI.) (2A). This correlates with afive-fold increase in G6PD activity (n=(2B). G6PD activity is expressedas units/6 min/mg protein and data are presented as mean±SEM. *p<0.01vs. 0×MOI. MOI multiplicity of infection.

FIGS. 3A-D show G6PD and endothelial cell migration. BAEC weretransfected with AS (3B) or the scrambled control, SS (3A), and observedto migrate from right to left across a vertical groove in the tissueculture plate. These representative frames demonstrate that VEGFincreases BAEC migration in SS (3C), but not AS (3D), transfected cells,suggesting that G6PD activity is important for cell migration.

FIG. 4 shows G6PD and cell migration. Fluorescently labeled, BAEC werestimulated with VEGF (100ng/ml) for 18 hr and cell migration wasassessed in a modified Boyden Chamber. In BAEC with normal G6PDactivity, VEGF significantly increased cell migration as determined byincreased fluorescence (13.3±2.6 vs. 55.3±6.6 units, p<0.01 vs.−VEGF,**p<0.01 vs.−DHEA.

FIGS. 5A and B show G6PD and Tube Formation. BAEC were transfected withan antisense oligodecocynucleotide to G6PD and mRNA (AS) or a scrambledcontrol (SS) and plated on Matrigel. Cells were then stimulated withVEGF and tube formation was assessed visually (5A) and quantified usingarea analysis with background substration (5B). Results of area analysisare the average of 10 high-powered fields.

FIGS. 6A and B show G6PD overexpression and tube formation. G6PDexpression was increased via adenoviral-mediated gene transfer. BAECwere then placed on Matrigel and stimulated with VEGF (100 nm/ml).

FIG. 7 shows the effect of VEGF on reactive oxygen species (ROS)production in BAEC.

FIG. 8 shows the effects of G6PD and NO on cell migration.

FIGS. 9A and B show a Matrigel plug from a WT mouse implanted 14 days at20×(9A) and 4×(9B) magnification stained with H&E. M=Matrigel,Mus=muscle layer, A=subcutaneous adipose tissue. Arrow shows cells thathave migrated into the Matrigel.

FIGS. 10A-D show G6PD and angiogenesis in vivo. H&E stain, 10×; WT=C3Hwild-type; HEMI=G6PD X^(b)Y.

FIGS. 11A and B show that cells migrating into the Matrigel plugs stainpositive for vWF antigen, demonstrating that they are endothelial cells.

FIGS. 12A-C show that cells migrating into the Matrigel plus stainpositive for eNOS and Nox1 antigens (arrows). 20× magnification.

FIGS. 13A-D show that G6PD gene transfer rescues G6PD deficientphenotype (HEMI mouse) and promotes migration into Matrigel. No addition(13A); VEGF (13B); AdG6PD, adenovirus encoding for murine G6PD (13C);AdG6PD and VEGF (13D). H&E stain. Panels A and C are 4× and panels B andD are 10× magnification.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis, the formation of new blood vessels in response to tissueischemia, is dependent upon a coordinated sequence of events involvingvascular endothelial cell migration, proliferation and tube formation.Vascular redox state, reactive oxygen species (ROS) formation, andnitric oxide (NO) have been shown to modulate growth factor-mediatedendothelial cell migration and proliferation. G6PD, the first andrate-limited enzyme in the pentose phosphate pathway that producesribose moieties for nucleic acid synthesis, is the principal cellularsource of NADPH. NADPH, in turn serves as a cofactor for enzymes thatregulate the cellular flux of ROS and endothelial nitric oxide synthase(eNOS) activity. Therefore, in that G6PD is involved in the productionof ROS and NO and thereby mediates endothelial cell proliferation andmigration to effect new vessel formation, it is a candidate to target tomodulate angiogenesis.

This discovery is important because of the role that angiogenesis playsin a variety of disease processes. On the other hand, where tissuesassociated with a disease condition require angiogenesis for tissuegrowth, it is desirable to inhibit angiogenesis and thereby inhibit thediseased tissue growth. Where injured tissue requires angiogenesis fortissue growth and healing, it is desirable to potentiate or promoteangiogenesis and thereby promote tissue healing and growth.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease tissue, inhibition ofangiogenesis will reduce the deleterious effects of the disease. Byinhibiting angiogenesis, one can intervene in the disease, amelioratethe symptoms, and in some cases cure the disease.

Examples of tissue associated with disease and neovascularization thatwill benefit from inhibitory modulation of angiogenesis include cancer,rheumatoid arthritis, ocular diseases such as diabetic retinopathy,inflammatory diseases, restenosis, and the like. Where the growth of newblood vessels is required to support growth of a deleterious tissue,inhibition of angiogenesis reduces the blood supply to the tissue andthereby contributes to reduction in tissue mass based on blood supplyrequirements. Particularly preferred examples include growth of tumorswhere neovascularization is a continual requirement in order that thetumor grow beyond a few millimeters in thickness, and for theestablishment of solid tumor metastases.

Where the growth of new blood vessels contributes to healing of tissue,potentiation of angiogenesis assists in healing. Examples includetreatment of patients with ischemic limbs in which there is abnormal,i.e. poor circulation as a result of diabetes or other conditions. Alsocontemplated are patients with chronic wounds which do not heal andtherefore could benefit from the increase in vascular cell proliferationand neovascularization.

A G6PD protein for use in the present invention can vary depending uponthe intended use. The terms “G6PD protein” or “G6PD” are used to refercollectively to the various forms of the enzyme, either in active orinactive forms.

An “active G6PD protein” refers to any of a variety of forms of the G6PDprotein, including peptides, which potentiate, stimulate, activate,induce or increase angiogenesis. Assays to measure potentiation ofangiogenesis are described herein, and are not to be construed aslimiting. A protein is considered active if the level of angiogenesis isat least 10% greater, preferably 25% greater, and more preferably 50%greater than a control level where no G6PD is added to the assay system.

An “inactive G6PD protein” or “G6PD antagonist” refers to any of avariety of forms of G6PD protein or other compound which inhibit,reduce, impede, or restrict angiogenesis or inhibit expression oractivity of G6PD. Assays to measure inhibition of angiogenesis aredescribed herein, and are not to be construed as limiting. A protein orcompound is considered inactive or an antagonist if the level ofangiogenesis is at least 10% lower, preferably 25% lower, and morepreferably 50% lower than a control level where no exogenous G6PD isadded to the assay system.

A G6PD protein useful in the present invention can be produced in any ofa variety of methods including isolation from natural sources includingtissue, production by recombinant DNA expression and purification, andthe like. G6PD protein can also be provided “in situ” by introduction ofa gene therapy system to the tissue of interest which then expresses theprotein in the tissue.

A gene encoding a G6PD protein can be prepared by a variety of methodsknown in the art. For example, the gene can readily be cloned using cDNAcloning methods from any tissue expressing the protein. The human cDNAaccession number is M21248 and the rat cDNA accession number isNM_(—)017006. Protein accession numbers are NP_(—)000393 andNP_(—)058702 for human and rat respectively.

The invention provides nucleotide sequences of particular use in thepresent invention. These define nucleic acid sequences which encode forG6PD protein useful in the invention, and various DNA segments,recombinant DNA (rDNA) molecules and vectors constructed for expressionof G6PD protein. DNA molecules (segments) of this invention thereforecan comprise sequences which encode whole structural genes, fragments ofstructural genes, and transcription units.

A preferred DNA segment is a nucleotide sequence which encodes a G6PDprotein as defined herein, or biologically active fragment thereof. Bybiologically active, it is meant that the expressed protein will have atleast some of the biological activity of the intact protein found in acell.

A preferred DNA segment codes for an amino acid residue sequencesubstantially the same as, and preferably consisting essentially of, anamino acid residue sequence or portions thereof corresponding to a G6PDprotein described herein.

A nucleic acid is any polynucleotide or nucleic acid fragment, whetherit be a polyribonucleotide of polydeoxyribonucleotide, i.e., RNA or DNA,or analogs thereof.

DNA segments are produced by a number of means including chemicalsynthesis methods and recombinant approaches, preferably by cloning orby polymerase chain reaction (PCR).

The G6PD gene of this invention can be cloned from a suitable source ofgenomic DNA or messenger RNA (mRNA) by a variety of biochemical methods.Cloning these genes can be conducted according to the general methodsknown in the art. Sources of nucleic acids for cloning a G6PD genesuitable for use in the methods of this invention can include genomicDNA or messenger RNA (mRNA) in the form of a cDNA library, from a tissuebelieved to express these proteins.

A preferred cloning method involves the preparation of a cDNA libraryusing standard methods, and isolating the G6PD-encoding or nucleotidesequence by PCR amplification using paired oligonucleotide primers basedon nucleotide sequences described herein. Alternatively, the desiredcDNA clones can be identified and isolated from a cDNA or genomiclibrary by conventional nucleic acid hybridization methods using ahybridization probe based on the nucleic acid sequences describedherein. Other methods of isolating and cloning suitable G6PD-encodingnucleic acids are readily apparent to one skilled in the art.

The invention also includes a recombinant DNA molecule (rDNA)containinga DNA segment encoding a G6PD as described herein. An expressible rDNAcan be produced by operatively (in frame, expressibly) linking a vectorto a G6PD encoding DNA segment of the present invention.

The choice of vector to which a DNA segment of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed. Both prokaryotic and eukaryotic expressionvectors are familiar to one of ordinary skill in the art of vectorconstruction, and are described by Sambrook et al., Molecular Cloning: ALaboratory Manual Cold Spring Harbor Laboratory (2001).

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to form the recombinantDNA molecules of the present invention. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of the desired DNA segment

Additionally, the angiogenesis modulator can also be delivered usinggene therapy. The gene transfer methods for gene therapy fall into threebroad categories: (1) physical (e.g., electroporation, direct genetransfer and particle bombardment), (2) chemical (e.g. lipid-basedcarriers and other non-viral vectors) and (3) biological (e.g. virusderived vectors). For example, non-viral vectors such as liposomescoated with DNA may be directly injected intravenously into the patientIt is believed that the liposome/DNA complexes are concentrated in theliver where they deliver the DNA to macrophages and Kupffer cells.

Gene therapy methodologies can also be described by delivery site.Fundamental ways to deliver genes include ex vivo gene transfer, in vivogene transfer, and in vitro gene transfer. In ex vivo gene transfer,cells are taken from the patient and grown in cell culture. The DNA istransfected into the cells, the transfected cells are expanded in numberand then reimplanted in the patient. In in vitro gene transfer, thetransformed cells are cells growing in culture, such as tissue culturecells, and not particular cells from a particular patient. These“laboratory cells” are transfected, the transfected cells are selectedand expanded for either implantation into a patient or for other uses.In vivo gene transfer involves introducing the DNA into the cells of thepatient when the cells are within the patient. All three of the broadbased categories described above may be used to achieve gene transfer invivo, ex vivo, and in vitro.

Mechanical (i.e. physical) methods of DNA delivery can be achieved bydirect injection of DNA, such as microinjection of DNA into germ orsomatic cells, pneumatically delivered DNA-coated particles, such as thegold particles used in a “gene gun,” and inorganic chemical approachessuch as calcium phosphate transfection. It has been found that physicalinjection of plasmid DNA into muscle cells yields a high percentage ofcells which are transfected and have a sustained expression of markergenes. The plasmid DNA may or may not integrate into the genome of thecells. Non-integration of the transfected DNA would allow thetransfection and expression of gene product proteins in terminallydifferentiated, non-proliferative tissues for a prolonged period of timewithout fear of mutational insertions, deletions, or alterations in thecellular or mitochondrial genome. Long-term, but not necessarilypermanent, transfer of therapeutic genes into specific cells may providetreatments for genetic diseases or for prophylactic use. The DNA couldbe reinjected periodically to maintain the gene product level withoutmutations occurring in the genomes of the recipient cells.Non-integration of exogenous DNAs may allow for the presence of severaldifferent exogenous DNA constructs within one cell with all of theconstructs expressing various gene products.

Particle-mediated gene transfer may also be employed for injecting DNAinto cells, tissues and organs. With a particle bombardment device, or“gene gun,” a motive force is generated to accelerate DNA-coated highdensity particles (such as gold or tungsten) to a high velocity thatallows penetration of the target organs, tissues or cells.Electroporation for gene transfer uses an electrical current to makecells or tissues susceptible to electroporation-mediated gene transfer.A brief electric impulse with a given field strength is used to increasethe permeability of a membrane in such a way that DNA molecules canpenetrate into the cells. The techniques of particle-mediated genetransfer and electroporation are well known to those of ordinary skillin the art.

Chemical methods of gene therapy involve carrier mediated gene transferthrough the use of fusogenic lipid vesicles such as liposomes or othervesicles for membrane fusion. A carrier harboring a DNA of interest canbe conveniently introduced into body fluids or the bloodstream and thensite specifically directed to the target organ or tissue in the body.Liposomes, for example, can be developed which are cell specific ororgan specific. The foreign DNA carried by the liposome thus will betaken up by those specific cells. Injection of immunoliposomes that aretargeted to a specific receptor on certain cells can be used as aconvenient method of inserting the DNA into the cells bearing thereceptor. Another carrier system that has been used is theasialoglycoprotein/polylysine conjugate system for carrying DNA tohepatocytes for in vivo gene transfer.

Transfected DNA may also be complexed with other kinds of carriers sothat the DNA is carried to the recipient cell and then resides in thecytoplasm or in the nucleoplasm of the recipient cell. DNA can becoupled to carrier nuclear proteins in specifically engineered vesiclecomplexes and carried directly into the nucleus.

Carrier mediated gene transfer may also involve the use of lipid-basedproteins which are not liposomes. For example, lipofectins andcytofectins are lipid-based positive ions that bind to negativelycharged DNA, forming a complex that can ferry the DNA across a cellmembrane. Fectins may also be used. Another method of carrier mediatedgene transfer involves receptor-based endocytosis. In this method, aligand (specific to a cell surface receptor) is made to form a complexwith a gene of interest and then injected into the bloodstream; targetcells that have the cell surface receptor will specifically bind theligand and transport the ligand-DNA complex into the cell.

Biological gene therapy methodologies usually employ viral vectors toinsert genes into cells. The term “vector” as used herein in the contextof biological gene therapy means a carrier that can contain or associatewith specific polynucleotide sequences and which functions to tansportthe specific polynucleotide sequences into a cell. The transfected cellsmay be cells derived from the patient's normal tissue, the patient'sdiseased tissue, or may be non-patient cells. Examples of vectorsinclude plasmids and infective microorganisms such as viruses, ornon-viral vectors such as the ligand-DNA conjugates, liposomes, andlipid-DNA complexes discussed above.

Viral vector systems which may be utilized in the present inventioninclude, but are not limited to, (a) adenovirus vectors; (b) retrovirusvectors; (c) adeno-associated virus vectors; (d) herpes simplex virusvectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) pox virus vectors such asan orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox orfowl pox; and (j) a helper-dependent or gutless adenovirus. In thepreferred embodiment the vector is an adenovirus.

Thus, a wide variety of gene transfer/gene therapy vectors andconstructs are known in the art. These vectors are readily adapted foruse in the methods of the present invention. By the appropriatemanipulation using recombinant DNA/molecular biology techniques toinsert an operatively linked G6PD encoding nucleic acid segment (eitheractive or inactive) into the selected expression/delivery vector, manyequivalent vectors for the practice of the present invention can begenerated.

Antagonists useful in the method of the present invention are G6DPantibodies, including monoclonal, chimeric humanized, and recombinantantibodies and fragment thereof which are characterized by high affinitybinding to G6DP in vivo and low toxicity. Neutralizing antibodies arereadily raised in animals such as rabbits or mice by immunization withG6DP. Immunized mice are particularly useful for providing sources of Bcells for the manufacture of hybridomas, which in turn are cultured toproduce large quantities of anti-G6DP monoclonal antibodies. Chimericantibodies are immunoglobin molecules characterized by two or moresegments or portions derived from different animal species. Generally,the variable region of the chimeric antibody is derived from a non-humanmammalian antibody, such as murine monoclonal antibody, and theimmunoglobin constant region is derived from a human immunoglobinmolecule. Preferably, both regions and the combination have lowimmunogenicity as routinely determined. Humanized antibodies areimmunoglobin molecules created by genetic engineering techniques inwhich the murine constant regions are replaced with human counterpartswhile retaining the murine antigen binding regions. The resultingmouse-human chimeric antibody should have reduced immunogenicity andimproved pharmacokinetics in humans. Preferred examples of high affinitymonoclonal antibodies and chimeric derivatives thereof, useful in themethods of the present invention, are described in the European PatentApplication EP 186,833; PCT Patent Application WO 92/16553; and U.S.Pat. No. 6,090,923.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi involvesmRNA degradation of a target gene. Results showed that RNAi isATP-dependent yet uncoupled from mRNA translation. That is, proteinsynthesis is not required for RNAi in vitro. In the RNAi reaction, bothstrands (sense and antisense) of the dsRNA are processed to small RNAfragments or segments of from about 21 to about 23 nucleotides (nt) inlength (RNAs with mobility in sequencing gels that correspond to markersthat are 21-23 nt in length, optionally referred to as 21-23 nt RNA).Processing of the dsRNA to the small RNA fragments does not require thetargeted mRNA, which demonstrates that the small RNA species isgenerated by processing of the dsRNA and not as a product ofdsRNA-targeted mRNA degradation. The mRNA is cleaved only within theregion of identity with the dsRNA. Cleavage occurs at sites 21-23nucleotides apart, the same interval observed for the dsRNA itself,suggesting that the 21-23 nucleotide fragments from the dsRNA areguiding mRNA cleavage. Isolated RNA molecules (double-stranded;single-stranded) of from about 21 to about 23 nucleotides mediate RNAi.That is, the isolated RNAs mediate degradation of mRNA of a gene towhich the mRNA corresponds (mediate degradation of mRNA that is thetranscriptional product of the gene, which is also referred to as atarget gene). Isolated RNA molecules specific to G6PD mRNA, whichmediate RNAi, are antagonists useful in the method of the presentinvention.

It will be appreciated by those of skill that cloned genes readily canbe manipulated to alter the amino acid sequence of a protein. The clonedgene for G6DP can be manipulated by a variety of well known techniquesfor in vitro mutagenesis, among others, to produce variants of thenaturally occurring human protein, herein referred to as muteins, thatmay be used in accordance with the invention.

The variation in primary structure of muteins of G6DP useful in theinvention, for instance, may include deletions, additions andsubstitutions. The substitutions may be conservative ornon-conservative. The differences between the natural protein and themutein generally conserve desired properties, mitigate or eliminateundesired properties and add desired or new properties.

Similarly, techniques for making small oligopeptides and polypeptidesthat exhibit activity of larger proteins from which they are derived (inprimary sequence) are well known and have become routine in the art.Thus, peptide analogs of proteins of the invention, such as peptideanalogs of G6DP that exhibit antagonist activity also are useful in theinvention.

Mimetics also can be used in accordance with the present invention tomodulate angiogenesis. The design of mimetics is known to those skilledin the art, and is generally understood to be peptides or otherrelatively small molecules that have an activity the same or similar tothat of a larger molecule, often a protein, on which they are modeled.

Variations and modifications to the above protein, inhibitors,antibodies and vectors to increase or decrease G6PD expression can belinked with a molecular counterligand for endothelial cell adhesionmolecules, such as PECAM-G6PD, to make these agents tissue specific.

In one aspect, the present invention provides for a method for themodulation of angiogenesis in a tissue associated with a disease processor condition, and thereby affect events in the tissue which depend uponangiogenesis. Generally, the method comprises administering to thetissue, associated with, or suffering from a disease process orcondition, an angiogenesis-modulating amount of a composition comprisinga G6PD protein or a nucleic acid vector expressing active or a G6PDantagonist.

Any of a variety of tissues, or organs comprised of organized tissues,can support angiogenesis in disease conditions including skin, muscle,gut, connective tissue, brain tissue, nerve cells, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

The patient to be treated according to the present invention in its manyembodiments is a human patient, although the invention is effective withrespect to all mammals. In this context, a “patient” is a human patientas well as a veterinary patient, a mammal of any mammalian species inwhich treatment of tissue associated with diseases involvingangiogenesis is desirable, particularly agricultural and domesticmammalian species.

Thus, the method embodying the present invention comprises administeringto a patient a therapeutically effective amount of a physiologicallytolerable composition containing a G6PD protein or nucleic acid vectorfor expressing a G6PD protein or an antagonist thereof.

The dosage ranges for the administration of a G6PD protein or antagonistdepend upon the form of the protein, and its potency, as describedfurther herein, and are amounts large enough to produce the desiredeffect in which angiogenesis and the disease symptoms mediated byangiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

A therapeutically effective amount is an amount of G6PD protein, ornucleic acid encoding for (active or inactive) G6PD protein or G6PDantagonist, sufficient to produce a measurable modulation ofangiogenesis in the tissue being treated, i.e., angiogenesis-modulatingamount. Modulation of angiogenesis can be measured or monitored by theCAM assay, examination of tumor tissues, or by other methods known toone skilled in the art.

The G6PD protein or nucleic acid vector expressing such protein or G6PDantagonist can be administered parenterally by injection or by gradualinfusion over time. Although the tissue to be treated can typically beaccessed in the body by systemic administration and therefore most oftentreated by intravenous administration of therapeutic compositions, othertissues and delivery means are contemplated where there is a likelihoodthat the tissue targeted contains the target molecule. Thus,compositions of the invention can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, and can be delivered by peristaltic means, if desired.

The therapeutic compositions containing a G6PD protein or nucleic acidvector expressing the protein or G6PD antagonist can be conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required physiologicallyacceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are peculiar to each individual.However, suitable dosage ranges for systemic application are disclosedherein and depend on the route of administration. Suitable regimes foradministration are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain concentrations inthe blood in the ranges specified for in vivo therapies arecontemplated.

G6PD protein, antagonists, and vectors may be adapted for catheter-baseddelivery systems including coated balloons, slow-release drug-elutingstents, microencapsulated PEG liposomes, or nanobeads for delivery usingdirect mechanical intervention with or without adjunctive techniquessuch as ultrasound.

There are a variety of diseases in which inhibition of angiogenesis isimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas and Kaposi sarcoma

Thus, methods which inhibit angiogenesis in a tissue associated with adisease condition ameliorates symptoms of the disease and, dependingupon the disease, can contribute to cure of the disease. In oneembodiment, the invention contemplates inhibition of angiogenesis, perse, in a tissue associated with a disease condition. The extent ofangiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods.

Thus, in one embodiment, a tissue to be treated is an inflamed tissueand the angiogenesis to be inhibited is inflamed tissue angiogenesiswhere there is neovascularization of inflamed tissue. This particularmethod includes inhibition of angiogenesis in arthritic tissues, such asin a patient with chronic articular rheumatism, in immune or non-immuneinflamed tissues, in psoriatic tissue, and the like.

In another embodiment, a tissue to be treated is a retinal tissue of apatient suffering from a retinal disease such as diabetic retinopathy orneovascular glaucoma and the angiogenesis to be inhibited is retinaltissue angiogenesis where there is neovascularization of retinal tissue.

In an additional embodiment, a tissue to be treated is a tumor tissue ofa patient with a solid tumor, a metastases, a skin cancer, a breastcancer, a hemangioma or angiofibroma and the like cancer, and theangiogenesis to be inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Typical solid tumor tissuestreatable by the present methods include lung, pancreas, breast, colon,laryngeal, ovarian, and the like tissues. Inhibition of tumor tissueangiogenesis is a particularly preferred embodiment because of theimportant role neovascularization plays in tumor growth. In the absenceof neovascularization of tumor tissue, the tumor tissue does not obtainthe required nutrients, slows in growth, ceases additional growth,regresses and ultimately becomes necrotic resulting in killing of thetumor.

Stated in other words, the present invention provides for a method ofinhibiting tumor neovascularization by inhibiting tumor angiogenesisaccording to the present methods. Similarly, the invention provides amethod of inhibiting tumor growth by practicing theangiogenesis-inhibiting methods.

The methods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

The G6PD antagonist of the invention may be combined with atherapeutically effective amount of an angiogenesis inhibiting factor,for example but not limited to, another molecule which negativelyregulates angiogenesis which may be, but is not limited to, plateletfactor 4, thrombospondin-1, tissue inhibitors of metalloproteases (TIMP1and TIMP2) prolactin (16-Kd fragment), angiostatin (38-Kd fragment ofplasminogen), bFGF soluble receptor, transforming growth factor-beta,interferon alfa, and placental proliferin-related protein.

In a yet further embodiment, the invention contemplates the practice ofthe method in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases as well as other forms of antiangiogenesistherapy. The administration of angiogenesis inhibitor is typicallyconducted during or after chemotherapy, although it is preferably toinhibit angiogenesis after a regimen of chemotherapy at times where thetumor tissue will be responding to the toxic assault by inducingangiogenesis to recover by the provision of a blood supply and nutrientsto the tumor tissue. In addition, it is preferred to administer theangiogenesis inhibition methods after surgery where solid tumors havebeen removed as a prophylaxis against metastases.

Insofar as the present methods apply to inhibition of tumorneovascularization, the methods can also apply to inhibition of tumortissue growth, to inhibition of tumor metastases formation, and toregression of established tumors.

Restenosis is a process of smooth muscle cell (SMC) migration andproliferation into the tissue at the site of percutaneous transluminalcoronary angioplasty which hampers the success of angioplasty. Themigration and proliferation of SMC's during restenosis can be considereda process of angiogenesis which is inhibited by the present methods.Therefore, the invention also contemplates inhibition of restenosis byinhibiting angiogenesis according to the present methods in a patientfollowing angioplasty procedures. For inhibition of restenosis, theinactivated G6PD is typically administered after the angioplastyprocedure because the coronary vessel wall is at risk of restenosis,typically for from about 2 to about 28 days, and more typically forabout the first 14 days following the procedure.

The present method for inhibiting angiogenesis in a tissue associatedwith a disease condition, and therefore for also practicing the methodsfor treatment of angiogenesis-related diseases, comprises contacting atissue in which angiogenesis is occurring, or is at risk for occurring,with a therapeutically effective amount of a composition comprising anG6PD antagonist.

In cases where it is desirable to promote or potentiate angiogenesis,administration of an active G6PD protein to the tissue is useful. Theroutes and timing of administration are comparable to the methodsdescribed above for inhibition. The G6PD protein may be used inconjunction with other angiogenesis promoting agents, e.g., VEGF orbFGF.

The present invention provides therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with a G6DP protein or vector capable ofexpressing a G6DP protein or G6PD antagonist as described herein,dissolved or dispersed therein as an active ingredient. In a preferredembodiment, the therapeutic composition is not immunogenic whenadministered to a mammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance theeffectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

For topical application, the G6PD antagonist may be combined with acarrier so that an effective dosage is delivered, based on the desiredactivity ie., ranging from an effective dosage, for example, about 1.0μM to 1.0 mM to prevent limitation, an ointment, cream, gel, paste,foam, aerosol, suppository, pad or gelled stick.

The amount of the active G6PD protein or G6PD antagonist (referred to as“agents”) used in the invention that will be effective in the treatmentof a particular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro assays such as those discussed hereinmay optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges foradministration of agents are generally about 0.01 pg/kg body weight to 1mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model testbioassays or systems.

Administration of the doses recited above can be repeated. In apreferred embodiment, the doses recited above are administered 2 to 7times per week. The duration of treatment depends upon the patient'sclinical progress and responsiveness to therapy.

The invention also contemplates an article of manufacture which is alabeled container for providing a G6DP protein or antagonist of theinvention. An article of manufacture comprises packaging material and apharmaceutical agent contained within the packaging material.

The pharmaceutical agent in an article of manufacture is any of thecompositions of the present invention suitable for providing a G6DPprotein or antagonist and formulated into a pharmaceutically acceptableform as described herein according to the disclosed indications. Thus,the composition can comprise a G6DP protein or a DNA molecule which iscapable of expressing the protein or an antagonist.

The article of manufacture contains an amount of pharmaceutical agentsufficient for use in treating a condition indicated herein, either inunit or multiple dosages.

The packaging material comprises a label which indicates the use of thepharmaceutical agent contained therein, e.g., for treating conditionsassisted by the inhibition or potentiation of angiogenesis, and the likeconditions disclosed herein.

The label can further include instructions for use and relatedinformation as may be required for marketing. The packaging material caninclude container(s) for storage of the pharmaceutical agent

As used herein, the term packaging material refers to a material such asglass, plastic, paper, foil, and the like capable of holding withinfixed means a pharmaceutical agent Thus, for example, the packagingmaterial can be plastic or glass vials, laminated envelopes and the likecontainers used to contain a pharmaceutical composition including thepharmaceutical agent.

In preferred embodiments, the packaging material includes a label thatis a tangible expression describing the contents of the article ofmanufacture and the use of the pharmaceutical agent contained therein.

The references cited throughout this application are herein incorporatedby reference.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications and publications cited herein are incorporated herein byreference.

EXAMPLE 1

Methodology to increase G6PD expression and activity include directinjection of G6PD DNA (“naked DNA”) and adenoviral-mediated genetransfer. We will use an adenoviral vector incorporating the cDNA forrat G6PD or cDNA for human G6PD, to increase G6PD expression. Toconstruct this vector, rat or human G6PD cDNA is excised from itsplasmid and inserted into a shuttle vector, pHIHG-Ad2. The resultingplasmid is then digested with Pac I and Mfe I and transferred into an E.coli BJ5183 strain together with a CIa I linearized adenoviral vector,pAd-hGM-CSF. This vector contains the entire adenoviral genomic sequenceexcept the E1 and E3 regions. Homologous recombination in the E. colistrain produced a recombinant adenoviral vector, pAd-G6PD, which wasextracted and transferred to E. coli DH5α for large-scale plasmidproduction. The sequence of the vector is confirmed by restrictiondigestion. PAD-G6PD is linearized with Pac I and transfected into 293cells using Lipofectamine® (Gibco). G6PD activity is measured andexpression confirmed by Western analysis. For full-scale preparation,following a 4-hr incubation at 37° C. with pAd-G6PD, transfected 293cells are placed in full growth media and harvested when the cellsbecome rounded due to viral cytopathic effect. The cell pellet is thenresuspended in glycerol 10% and recombinant virus will be extracted byrepeated freeze-thaw.

EXAMPLE 2

G6PD and Endothelial Cell Proliferation

To investigate the role of G6PD in vascular endothelial cellproliferation, we initially sought to determine if growth factorsinvolved in angiogenesis, such as vascular endothelial growth factor(VEGF), influenced G6PD activity. To examine this effect, we treatedbovine aortic endothelial cells (BAEC) with vascular endothelial growthfactor (VEGF)(100 ng/ml), an essential growth factor for angiogenesis,and, compared to untreated cells, demonstrated a significant increase inG6PD activity after 4 hr (60.9±18 vs. 182.7±37 units/6 min/mg protein,p<0.01, n=6), that returned toward baseline by 24 hr (92.0±22 vs. 87±19units/6 min/mg protein, p=NS, n-6). Increased BAEC G6PD activityfollowing stimulation with VEGF was paralleled by an increase incellular NADPH levels at 4 hr (0.3±0.02 vs. 0.5±0.04 mmoles/mg protein,p<0.01, n—3), which similarly returned toward baseline at 24 hr(0.3±0.08 vs. 0.4±0.08 mmoles/mg protein, p=NS, n=3). Western analysisrevealed that VEGF did not increase G6PD expression at either time point(data not shown). These observations suggested that VEGF increased basalG6PD activity to enhance NADPH levels, and did not stimulate de novosynthesis of G6PD.

To examine further the role of G6PD in VEGF-mediated cell proliferation,we next inhibited G6PD activity by exposing BAEC todehydroepiandrosterone (DHEA) (100 pmol/L), a noncompetitive inhibitorof G6PD, for 24 hr. We have previously demonstrated that DHEA-treatedBAEC have a 60% reduction in G6PD activity resulting in a 30% decreasein the level of cellular NADPH. Compared to control cells, DHEA-treatedBAEC demonstrated a significant decrease in [³H]-thymidine incorporationunder basal conditions (31,125±2,410 vs. 9,967±1,531 cpm, p<0.01, n=4),and following stimulation with VEGF (100 ng/ml for 8 hr) (39,223±7,158vs. 8,902±1,451 cpm, p<0.01, n=4) (FIG. 1A). These results were not theresult of increased cell death as determined by lactate dehydrogenaseactivity measured in the media

To confirm these results, G6PD expression was inhibited in BAEC bytransfection with an antisense phosphorothioate oligodeoxynucleotide toG6PD mRNA (5′-AGGUCACCCGAUGCACCCAUGAUGA-3′ (SEQ ID NO: 1)) (AS) usingOligofectin I (Sequitor, Inc.) as a vehicle. As a control, we used aphosphorothioate oligodeoxynucleotide with a scrambled sequence (SS). Wehave previously demonstrated that following transfection for 5 hours,Western analysis revealed a significant decrease in G6PD expression thatwas associated with a 50% reduction in G6PD activity and a 40% decreasein NADPH levels. Compared to SS-transfected BAEC, AS-transfected BAEC,with decreased G6PD expression, demonstrated decreased basal(35,258±2,368 vs. 21,109±3,284 cpm, p<0.01, n=3) and VEGF-stimulated(56,038±2,775 vs. 30,667±3,361 cpm, p<0.01, n=3):cell proliferation(FIG. 1B).

We have recently constructed an adenoviral vector incorporating murineG6PD cDNA (93% homologous to the rat cDNA). The sequence of this vector,pAd-G6PD, was confirmed by restriction digestion, and expression of theactive enzyme was demonstrated in E. coli lysates. We have successfullytrans(in)fected BAEC and, compared to cells trans(in)fected with emptyvector, shown a significant increase in G6PD expression, activity(108.3±6.7 vs. 571.2±30.8 units/6 min/mg protein, p<0.01, n=6) (FIG. 2),and cellular NADPH levels (0.5±0.01 vs. 0.67±0.02 mmoles/mg protein,p<0.01, n=3). Gene transfer of G6PD resulted in a significant increasein cell proliferation as determined by [³H]-thymidine incorporation(31,983±3,009 vs. 51,769±2,784 cpm, p<0.01, n=3).

G6PD and Tube Formation

To examine the role of G6PD in endothelial cell tube formation in vitro,we plated BAEC on Matrigel and stimulated cells with VEGF (100ng/ml) for12 hr. Results were quantified using NIH Image to determine area of tubeformation. Under basal conditions, BAEC plated on Matrigel formed tubesand networks that were increased following exposure to VEGF.VEGF-mediated tube formation was significantly decreased inAs-transfected BAEC compared to cells transfected with a scrambledcontrol, and this correlated with a significant decrease in vessel area(FIG. 5). Similar results were obtained for DHEA-treated BAEC,demonstrating that G6PD importantly modulates endothelial cell tubeformation.

In order to confirm that G6PD importantly modulates tube formation invitro, we next increased G6PD expression via adenoviral-mediated genetransfer and examined tube formation. Increased G6PD expressionsignificantly increased endothelial cell tube under basal conditions aswell as following VEGF stimulation (FIG. 6).

G6PD and Reactive Oxygen Species

As growth factors have been suggested to increase cell proliferation andmigration via the generation of reactive oxygen species (ROS), we nextexamined the effect of VEGF on ROS production in BAEC using2′,7′-dichlorofluorescein diacetate fluorescence. BAEC were exposed toVEGF (100 ng/ml) for 4 hr, after which ROS formation was measured every15 min over the course of 1 hr. At 1 hr., BAEC stimulated with VEGFdemonstrated an increase in ROS formation compared to untreated cells(61.8±101.5 units, p<0.01, n=8), and, interestingly, this response wasabrogated in AS-transfected BAEC (299±68.9 units, p<0.05, n=8) (FIG. 7).These observations suggested that ROS formation may play an importantrole in VEGF-mediated cell proliferation and deficient G6PD activityinfluences this response.

G6PD and Endothelial Cell Migration

We next examined the role of G6PD on VEGF-stimulated endothelial cellmigration. In a cell-wounding migration assay, BAEC were grown toconfluence on a P100 culture dish. Using a sterile scalpel, a verticalincision was made in the midline of the dish, and under microscopicguidance, BAEC were removed from one-half of the plate. BAEC were thenincubated with VEGF (100 ng/ml) in serum-free media, and cell migrationacross the midline was observed after 18 hr and quantified by theaverage cell count in 5 high powered fields. VEGF significantlyincreased BAEC migration (14±4 vs. 28±6 cells/hpf, p<0.01, n=5), aneffect that was significantly decreased in BAEC pretreated with DHEAcompared to untreated cells (38.6±6 vs. 10±2 cells/hpf, p<0.01, n=5) ortransfected with AS compared to SS-transfected cells (52±11 vs. 16±5cells/hpf, p<0.01, n=5). A representative cell-wounding migration assayin BAEC transfected with SS or AS is shown in FIG. 3.

To confirm these observations, we analyzed cell migration using amodified Boyden chamber. BAEC were fluorescently labeled with calcein AM(Molecular Probes, Inc.) and 0.4×10³ cells were loaded to the top of apre-fitted membrane filter that fit a specially designed 96-wellmicroplate. The microplate wells were loaded with serum-free media andVEGF (100 ng/ml) for 18 hr., and cells were allowed to migrate acrossthe membrane. The plate was then analyzed using a fluorescent microplatereader. In BAEC treated with VEGF, there was a significant increase influorescence, indicating an increase in cell migration, that wasabrogated in DHEA-treated BAEC (FIG. 4).

G6PD and Nitric Oxide

Nitric oxide has been implicated as a second messenger important forendothelial cell proliferation and migration. We have previouslyestablished that G6PD modulates NO levels, and that decreased G6PDactivity or expression is associated with a reduction in bioavailableNO. To determine if increased G6PD activity would restore bioavailableNO levels, we transfected BAEC with pAdG6PD and examined markers of NOproduction. Nitric oxide bioactivity was increased in AdG6PD BAEC asdemonstrated by cGMP production (1.8±0.1 vs. 2.7±0.3 pmol cGMP/mgprotein, p<0.04) and nitrate production (865.91±93.3 vs. 1232.2±221.2pmol nitrate/mg protein, p<0.001) in response to bradykinin. Theseobservations suggested that G6PD and eNOS activity function in parallel,and owing to the absolute requirement of NADPH by eNOS, we hypothesizedthat G6PD and eNOS may colocalize.

To examine this hypothesis, BAEC were grown to confluence on slides andstimulated with A23187 (5 μmol/L) for 10 min. Cells were then exposed toanti-G6PD and anti-eNOS antibodies, and blocked with fluorescein andrhodamine secondary antibodies. Cells were visualized using confocalmicroscopy.

To examine the functional significance of G6PD and NO with respect tocell migration, DHEA-treated BAEC were stimulated with VEGF (100/ng/ml)for 18 her., in the presence of absence of eNOS inhibitors, L-Name (1mmol/L), which inhibits both NO and ROS formation by eNOS, and —NMMA(100 μmol/L), which only inhibits NO formation. Migration was assayedusing a modified Boyden chamber as described above (FIG. 8). Inhibitionof eNOS with L-NAME (55.3±16.6 vs. 23.6±17 units, p<0.05, n=6) or L-NMMA(55.3±16.6 vs. 21.6±7.3 units, p<0.04, n-6) significantly decreased cellmigration in VEGF-stimulated cells compared to control cells.DHEA-treated BAEC, VEGF did not significantly increase migration.

EXAMPLE 3

G6PD-Deficient Murine Model

All efforts to create a G6PD knockout mouse have been unsuccessful owingto the embryonic lethality of deletion of this gene; however, thePretsch mouse, a murine model of G6PD deficiency, demonstratessignificantly reduced G6PD expression and activity. G6PD is an X-linkedgene, and therefore, females may be either homozygous or heterozygousfor the mutation while male offspring are hemizygous. This mutation isstably transmitted to offspring, and, through direct sequencing ofPCR-amplified genomic DNA, and comparison with genomic DNA from othermouse strains, a single base difference (A-T to T-A) was identified inexon 1. We have developed G6PD mouse primers and are able tosuccessfully genotype our animals. In addition, we have correlated theG6PD deficient genotype with a G6PD deficient phenotype in hepaticlysates. Compared to C3H wild-type mice with 100% G6PD activity,homozygous female mice have 31% activity, heterozygous female mice have52% activity, and hemizygous male mice have 23% activity. We measuredNADPH levels in hemizygous male mice and found a 50% decrease in NADPHlevels in compared to wild-type mice. These observations confirm thatthe Pretsch mouse is a reliable model of G6PD deficiency that may beeasily genotyped and is phenotypically characterized by reduced G6PDactivity and decreased NADPH levels.

To examine the effect of G6PD activity on angiogenesis in vivo, weperformed an in vivo Matrigel migration assay in C3H wild-type (WT) andhemizygous male (HEMI) mice. Matrigel, derived from mouse basementmembrane proteins, was implanted subcutaneously in the groin area toform a Matrigel “plug”. Each mouse was injected on one side with eitherMatrigel alone, or Matrigel supplemented with VEGF (100 ng/ml). In thismanner, each animal could serve as its own control. Plugs were left inplace for 14 days. Following this time, the animals were euthanized,plugs excised with surrounding tissue for orientation, and embedded andsectioned in paraffin. Sections were then stained with Hematoxylin &Eosin (H&E). Images were captured digitally and assessed for cellcounts. For orientation purposes, a representative Matrigel plug from aWT mouse at 4× (right) and 20× (left) magnification is shown below (FIG.9).

We next implanted Matrigel plugs for 14 days in WT and HEMI mice todetermine the effect of G6PD on angiogenesis in vivo (FIG. 10). In WTmice, there is noticeable cell migration into the Matrigel plug (upperleft), and this response is increased in Matrigel supplemented with VEGF(upper right). In contrast, in HEMI mice, there is a marked decrease incell migration into the Matrigel plug compared to WT mice (bottom left),and exposure to VEGF did not significantly improve this response.

These findings were quantified by counting cells in 10 randomly selectedhigh-power (20×) fields per image in 3 successive images. In WT mice,VEGF increased the number of cells migrating into the Matrigel plug;however, in HEMI mice, there was a marked reduction of cell migrationinto the Matrigel alone, or supplemented with VEGF. These findingsconfirm that G6PD modulates angiogenesis in vivo.

We next wanted to determine that the cells that migrated into theMatrigel were endothelial cells. Therefore, we used immunohistochemistrytechniques with antigen retrieval to stain the Matrigel sections for theendothelial cell markers CD31 and von Willebrand factor (vWF) (FIG. 11).Serial sections of a Matrigel plug obtained from a WT mouse were stainedwith H&E (left), or for vWF antigen (right) and visualized at 20×. Thesefindings demonstrate that the cells migrating into the Matrigel plugsare endothelial cells. Similar findings were obtained when cells werestained for CD31.

We performed further immunohistochemical analysis with antigen retrievalof serial sections of a Matrigel plug obtained from a WT mouse toconfirm the presence of eNOS antigen as well as Nox1 (homologous to thecatalytic subunit gp91phox of NADPH oxidase) antigen (FIG. 12). In thisstudy, cells were initially stained for CD31 and vWF antigen todemonstrate that they were endothelial cells. These cells additionallystained positive for eNOS and Nox1 antigens as shown below.

We next wanted to determine if we could “rescue” the G6PD-deficientphenotype using gene transfer to increase local G6PD expression. Wetreated Matrigel with an adenoviral vector encoding for G6PD implantedplugs in HEMI mice for 7 days in the presence or absence of VEGF (FIG.13). In HEMI mice, there was minimal cell migration into the Matrigelplug (Panel A) that was not increased significantly by VEGF stimulation(Panel B). In contrast, local gene transfer of G6PD in HEMI miceresulted in a marked increase in migrating cells into the Matrigel plug(Panel C) and this effect was enhanced further by VEGF (Panel D).

Our studies demonstrate that in a murine model of G6PD deficiency wehave established an in vivo measure of angiogenesis, the Matrigel plugassay and conformed that G6PD regulates angiogenesis in this model.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

REFERENCES

The following references and all others cited in the specification areincorporated by reference.

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1. A method for stimulating angiogenesis in a tissue associated with adisease condition comprising administering to said tissue anangiogenesis stimulating amount of a pharmaceutical compositioncomprising a G6PD protein or a nucleotide sequence encoding for saidprotein.
 2. The method of claim 1 wherein said tissue has abnormalcirculation.
 3. A method for inhibiting angiogenesis in a tissueassociated with a disease condition comprising administering to saidtissue an angiogenesis inhibiting amount of a pharmaceutical compositioncomprising a G6PD antagonist.
 4. The method of claim 3 wherein saidtissue is a solid tumor or solid tumor metastasis.
 5. The method ofclaim 3, wherein said administering is conducted in conjunction withchemotherapy.
 6. The method of claim 3, wherein said tissue is retinaltissue and said condition is retinopathy, diabetic retinopathy ormacular degeneration.
 7. The method of claim 3, wherein said tissue isat the site of coronary angioplasty and said tissue is at risk forrestenosis.
 8. The method of claim 1 or 3, wherein said administeringcomprises intravenous, transdermal, intrasynovial, intramuscular, ororal administration.
 9. The method of claim 3, wherein said tissue isinflamed and said condition is arthritis or rheumatoid arthritis.
 10. Apharmaceutical composition for stimulating angiogenesis in a targetmammalian tissue comprising a gene transfer vector containing a nucleicacid, said nucleic acid having a nucleic acid segment encoding for aG6PD protein and a pharmaceutically acceptable carrier or excipient. 11.A pharmaceutical composition for inhibiting angiogenesis in a targetmammalian tissue comprising a gene transfer vector containing a nucleicacid, said nucleic acid having a nucleic acid segment encoding aantisense G6PD oligonucleotide and a pharmaceutically acceptable carrieror excipient.
 12. A pharmaceutical composition for stimulatingangiogenesis in a target mammalian tissue comprising a therapeuticamount of a G6PD protein, and a pharmaceutically acceptable carrier orexcipient.
 13. A pharmaceutical composition for inhibiting angiogenesisin a target mammalian tissue comprising a therapeutic amount of a G6PDantagonist, and a pharmaceutically acceptable carrier or excipient. 14.An article of manufacture comprising packaging material and apharmaceutical composition contained within said packaging material,wherein said pharmaceutical composition is capable of inhibitingangiogenesis in a tissue associated with a disease condition, whereinsaid packaging material comprises a label which indicates that saidpharmaceutical composition can be used for treating disease conditionsby inhibiting angiogenesis, and wherein said pharmaceutical compositioncomprises a G6PD antagonist.
 15. An article of manufacture comprisingpackaging material and a pharmaceutical composition contained withinsaid packaging material, wherein said pharmaceutical composition iscapable of stimulating angiogenesis in a tissue associated with adisease condition, wherein said packaging material comprises a labelwhich indicates that said pharmaceutical composition can be used fortreating disease conditions by stimulating angiogenesis, and whereinsaid pharmaceutical composition comprises a G6PD protein or a vectorcontaining a DNA segment encoding said protein.